PROJECT SUMMARY Cellular development is controlled by cell surface proteoglycans, a class of glycoconjugates that can orchestrate the binding and release of growth factors to initiate differentiation or prime stem cells towards specific lineages. Proteoglycans are attractive targets for exerting exogenous control to re-program cellular differentiation, and yet despite their potential for biomedicine, strategies that harness their regulatory functions are limited. This gap is caused by the lack of techniques that recognize their chemical and nanoscale complexity. Proteoglycans are composed of a core protein covalently linked to a highly complex, heterogenous mixture of sulfated glycosaminoglycan carbohydrates. Furthermore, proteoglycans can be located in distinct compartments of the cell membrane, and this distribution can lead to different outcomes in differentiation. This application outlines the development of chemical strategies to mimic and probe the nanoscale architecture and organization of proteoglycans in cellular development. In two specific aims, we will demonstrate that the techniques developed through this proposal will enable new chemical strategies to control stem cell differentiation and the regeneration of skeletal muscles. First, we will develop protein engineering strategies that facilitate the bioorthogonal conjugation of semi-synthetic glycosaminoglycans to specified sites along the proteoglycan core protein chain, in order to generate engineered cell surface proteoglycans with pre-mapped glycosylation sites, as well as defined glycosaminoglycan compositions and lengths. Second, we will develop chemical methods to direct and monitor the membrane localization of such engineered proteoglycans. Both strategies will be applied in order to study and to exert control over the differentiation of mouse embryonic stem cells and the activation of skeletal satellite muscle cells. The long-term goal of this proposal is to offer strategies to alter proteoglycan structure in order to manipulate cellular differentiation, and to harness this knowledge towards understanding the factors that regulate canonical human development.